The present invention relates to a method and apparatus for activating a thermo-enzyme reaction by applying electromagnetic energy to a target to produce a rapid elevation in the temperature of at least a portion of the target.
The present invention is applicable to certain temperature-sensitive transformations of biological systems. For example, the present invention is applicable to the temperature-sensitive transformation of cells with the specific synthesis or activation of bio-molecules such as enzymes and/or structural heat shock proteins. A number of thermally-induced reactions that in turn activate or regulate other enzyme activities in biological systems are of importance for the induction of various molecular syntheses or molecular processings. Some genetic mutations may be revealed following temperature changes of a few degrees centigrade which result in the production of new structural proteins that participate in the membranous assembly of a cell and therefore can be used for diagnostic purposes. Some heat shock proteins are specific to cancer or to infectious pathogens and constitute valuable markers capable of eliciting immune responses. Heat shock protein syntheses or processings can be made to occur in vitro in experimental systems, in living cells and tissues, or in intact organisms. Typically, specimens are exposed to non-lethal temperature variations from 1 to 20.degree. C. circa the optimum temperature conditions for the observed specimens (i.e. 37.degree. C. for specimen of human origin) for periods from several seconds to several hours duration. The ability to report the protein products using a variety of biochemical, histological, and/or microscopy techniques, allows for the identification of specific heat induced markers. These markers can be used for diagnostically detecting genetic mutations, such as, but not limited to, cancer and other inheritable diseases. Identified markers are also useful to assist in developing techniques for gene therapy, and to diagnose the presence of pathogens responsible for a variety of diseases including, but not limited to, HIV, malaria, hepatitis, and other bacteria-mediated diseases. Nonetheless, current methodologies for experimental thermal induction of biological reactions have limits. These reactions are typically induced by mass temperature increases or decreases that affect entire populations of cells, large tissue specimens, or even intact live animals. By contrast, the present invention teaches a new method for temperature control using the property of energy transfer from a radiative source of infrared light directed to at least a portion of a microscopic target or to single cells.
The present invention is particularly applicable to thermo-enzyme cycling reactions, especially the amplification of nucleic acids into polynucleotides using polymerase chain reaction (PCR).
Polymerase chain reaction (PCR) is a method for the in vitro synthesis of nucleic acids. A particular segment of DNA or RNA is specifically extended or replicated in vitro during a repeated and thermally controlled enzymatic reaction. Successive cycles of amplification, which refers to the accumulation of identical copies of a nucleic acid template by repetitive duplication of the template and its copies, and encompasses the three phases of denaturation, annealing, and extension, double the amount of the target DNA synthesized in the previous cycle. Procedures are well established and a significant product yield is possible even when starting with an extremely small amount of template.
Applications for PCR technologies are found in various domains of research and diagnostics, including sequencing or mutagenesis of genetic material, analysis and diagnosis of genetic disorders and diseases, forensics and evolutionary investigations. The societal benefits of PCR have been well documented with respect to quality testing and for various diagnoses, for example with the detection of microorganisms such as bacteria and viruses in water supplies and with the early detection of cancer or of diseases.
Optimization of PCR reactions requires decisive control over several parameters. The occurrence of artifacts is limited with properly defined concentration requirements for enzyme, molecular reagents including template sequence, salts, and for the buffer solution. Most importantly, precise control over the temperature of the reaction during its entire duration is critical to the successful production of highly specific products. Repeated cycles of heating and cooling are necessary for the complete denaturation of the template molecules, for the reliable annealing of the primers to their complementary sequences and for their correct extension with the action of the DNA polymerase. It is also important to select a reaction vessel with fast thermal transmission characteristics and minimum thermal capacity to avoid absorption from the heat source. Typical denaturation conditions vary from 94.degree. C. to 99.degree. C. and between from a few seconds to a minute. Typical annealing conditions vary between 55.degree. C. and 75.degree. C. and require from a few seconds to one minute to complete. Optimum annealing temperature must be determined precisely to assure best product yield. The correct extension of the primers also depends upon the temperature of the reaction and is usually performed between about 70.degree. C. and 75.degree. C.
DNA amplification can be performed directly in individual cells and tissue sections on a microscope slide. In situ PCR makes the detection of specific nucleic acid sequences highly sensitive and fast. Current methods involve the thermal cycling of the entire slide specimen that is placed in contact with a thermocycler's heater block. Evaporation control devices must be added to the slide, such as a layer of wax or a self-adhesive chamber device. Typically the entire specimen is exposed to the thermal cycling. In contrast, the present invention proposes to limit the thermal cycling phenomenon to the cell chromosome or target of interest by the irradiation profile of a controlled source of electromagnetic energy.
During the entire duration of the amplification process, different precise temperature levels must be rapidly reached and stabilized for well defined periods of time in a cyclical manner for the denaturation, annealing and extension phases. To achieve this, commercially available automated instruments known as thermocyclers are preferred to less accurate manual methods in which reaction vessels containing the reagents are systematically transferred between water baths that are maintained at different temperatures. The technical requirements for automated or semi-automated instruments include a regulated heater or heat source with associated monitoring device, a timer, a reaction chamber and possibly a programmable controller. The reaction vessels have fast thermal transmission characteristics and minimal thermal capacity. Using micro-capillary tubes to contain the reaction or thin walled microcentrifuge tubes with a high surface area to volume ratio cuts sample temperature lag. Temperature elevation is obtained via conduction or convection phenomena. In the conduction design, the reaction vessels are placed in direct contact with the heater element, a conventional design of which includes a single heat block with integrated cooling provided by circulation of refrigerant or of tap water. In the convection design, heat transfer to the reaction vessels is via a fluid such as oil, a polymer or simply a layer of air. The combination of high velocity air as the heating and cooling medium and low thermal inertia vessels ensures both temperature uniformity and rapid heat exchange with the sample, which benefits specificity of the reaction.
Both conduction and convection methodologies have limits and for example are inappropriate to permit PCR on a specimen under inspection using microscopy. In contrast, the present invention provides a new method for temperature control using the property of energy transfer from a controlled radiative source of electromagnetic energy which is amenable to microscopy applications. Electromagnetic radiations refer to a range of wavelengths or frequencies that are propagated by simultaneous periodic variations of electric and magnetic field intensity and that include gamma rays, X rays, ultraviolet, visible light, infra red, and radio waves to the longest radio waves. The present invention teaches the use of controlled preferably infrared energy extending from 750 nm to millimeter waves to produce molecular effects in biological specimens and/or biological systems. For example, one or more infrared laser beams provide a suitable source of controlled electromagnetic energy for the invention. Other light sources are possible alternatives, including one or more lamps, such as a filament lamp, an encased gas illuminator, and a pressurized gas lamp or electrical arc lamp that uses gas discharge to produce either a continuous or a discrete light spectrum that includes infra red waves.
Photothermal properties of laser light applied to promote temperature elevation of media and substrates are also known (Welch, A. J. et al., "Laser thermal ablation", Photochemistry and Photobiology, 53, 1991, 815-823). Laser thermal effects are used for ablation of various surfaces or tissues, with principal applications in medical sciences, such as dermatology treatments. Although technically attainable with either continuous wave or pulsed laser sources, laser ablation of tissues in medical applications is essentially reported for pulsed laser geometries which offer greater control over irradiation and permit minimized heat losses due to conduction. Water content of the target substrates plays a major role in the successful transfer of energy in the case of infrared laser sources. In the IR range of the electromagnetic spectrum, for wavelengths above 1300 nm, water, OH and amines are the dominant chromophores, and high contents in water clearly affects the local rate of heat generation at these wavelengths. Furthermore, the presence of water, or enhanced water content due to hydration, increases thermal conductivity and therefore heat conduction. Lasers that emit infrared light, for example at a wavelength of 10600 nm such as produced by a CO.sup.2 laser, at a wavelength of 2940 nm from an Er:YAG laser, at 2100 nm from an Ho:YAG laser, at 1480 nm using a solid state diode laser, at a wavelength of either 1320 nm or 1064 nm from an Nd:YAG laser, and other lasers that emit within these boundaries of the spectrum have therefore been utilized for laser thermal applications. Technical requirements for medical laser-based instruments are principally established with reference to the penetration depth of the beam and its fluence. Since the penetration depth in the infrared range of the spectrum is determined by the relative absorption in water, laser wavelengths that coincide with an absorption peak of water constitute optimal choices.
Light energy and in particular laser energy in the field of microscopy is applied in a wide variety of applications. Laser beams are being used in combination with microscopes for the purpose of illuminating microscopic specimens or to produce specific effects. Typically, the beam is directed towards the back aperture of the objective lens which focuses the beam onto a localized area of the specimen. The same objective lens can be used to simultaneously observe the specimen. Precise positioning and alignment of the laser beam in multiple planes relative to the optical path of the microscope are made possible with adjustable lenses and mirrors. Laser sources are also coupled to microscopes via a flexible optical fiber that serves as a light guide.
Lasers coupled to microscopes are used as microscopy tools, e.g., for optical trapping and laser microablation. Manipulation of microscopic structures is discussed in U.S. Pat. Nos. 4,893,886; 5,170,890; and 5,283,417. In general, these patents discuss methods for trapping microscopic particles in a photon gradient, or for controllably damaging microscopic structures with laser energy. The ablative properties of laser microscope devices find applications in biology (Berns, M. W., et al., "Laser microsurgery in cell and development biology", Science, 213, 1981, 505-513; Rink, .et al, "1.48 .mu.m Diode Laser Microdissection of the Zona Pellucida of Mouse Zygotes", Proceedings SPIE, 2134A, 412-422). U.S. Pat. No.4,629,687 describes a method and apparatus for the positive selection of cells using a focused laser beam to kill unwanted cells. A technique called "Laser Capture Microdissection" includes picking out specific cells in vitro from a thin tissue section placed on a glass microscope slide by thermally modifying adhesive properties of a substrate (Emmert-Buck, M. R. et al., "Laser capture microdissection", Science, 274, 1996, 998-1001). Other applications describe the use of a microscope and laser assembly containing wavelength-selective mirrors for directing externally produced light into a microscopic sample for the purpose of stimulating fluorescence in the sample. With confocal microscopy, elaborate scanning illumination of a specimen is performed by a tightly focused spot of light (Matsumoto, B. and ramer, T., "Theory and applications of confocal microscopy", Cell Vision, 1, 1994, 190-198). Microscope and laser assembly devices are used to produce multiple photon excitation which involves the simultaneous absorption of two, three or eventually more laser photons by the same fluorophore. Multiple photon excitation techniques allow fluorescence emission to occur at a wavelength substantially shorter than the excitation and allow high resolution imaging without a confocal aperture.
A review of the literature demonstrates the usefulness of laser-microscopy technologies. A laser beam focused by the objective lens of a microscope can be used to illuminate or to transfer energy to a very small part of a microscopic target specimen. Although a variety of methods and devices have been described for both research and medical applications, the benefit of using a laser beam as a means to produce substantial energy delivery and thus temperature elevation, has not yet been investigated for applications with reference to the activation of thermo-enzyme reactions and in particular with reference to the amplification of nucleic acids with polymerase chain reaction. The prior art teaches and suggests the use of laser beams only for the destruction or ablation of cells, and at best provides means to limit damage to adjoining cells, which is essentially due to heat conduction. Thus, heretofore known devices disclose minimum heat deposition during short irradiation exposure times for ablation processes. However, it is an object of the present invention to be able to apply electromagnetic energy, such as a laser beam, to a thermo-enzyme reaction such as a polymerase chain reaction to purposely produce a sustained temperature elevation of a target specimen without ablation. In particular, we have unexpectedly found that the temperature elevation that can be anticipated from using a laser source in the infrared range of the spectrum is well within the boundaries applicable to produce certain temperature-sensitive transformation of biological systems such as with activation of enzymes or heat shock proteins or with PCR reactions.